Figure 4.

USP1 deubiquitinates KDM4A. A, 293T cells were transfected with Flag‐KDM4A, Myc‐USP1, and HA‐tagged ubiquitin (HA‐ub) as indicated. Cellular lysates were coimmunoprecipitated by anti‐Flag Ab. Polyubiquitylated KDM4A protein was detected by anti‐HA‐ub Ab. B, RV1 cells stably expressing control or USP1 shRNAs were subjected to ubiquitination assay and the polyubiquitylated KDM4A protein was detected by anti‐ub Ab. C, RV1 cells transfected with Flag‐KDM4A were treated with or without 10 μmol/L ML323 (USP1 inhibitor). Polyubiquitylated KDM4A protein was examined as in (B). D, Deubiquitination of KDM4A in vitro by USP1. Ubiquitinated KDM4A was incubated with purified GST fusion USP1 (WT) or USP39 (CS) in vitro and then blotted with indicated Abs. E, 293T cells were transfected with Myc‐USP1, Flag‐KDM4A, and HA‐ub (WT, K6R mutant, K11 mutant, K27 mutant, K29 mutant, K33 mutant, K48 mutant, and K63 mutant). Analysis was carried out as described in (A)